At least some of the present invention may have been made with funds from the United States Government, which may therefore have certain rights in this invention.
1. Field of the Invention
Aspects of the present invention relate to the discovery and use of baculovirus RNA polymerase for the production of capped and polyadenylated transcripts in vivo and in vitro. Aspects relate to useful tools and techniques for biotechnologists, including more stable RNA transcripts produced.
2. Background of the Related Art
A. Baculovirus
Baculoviruses are popular eukaryotic expression vectors. The baculovirus system has been used to produce hundreds of different proteins for basic research and pharmaceutical applications such as medical therapeutics, diagnostics, vaccines, and drug discovery. Baculoviruses have been adapted as expression vectors because they normally produce abundant amounts of a viral protein called polyhedrin during the very late stage of viral infection. Polyhedrin is essential for the propagation of baculoviruses in insects, but is nonessential for growth in tissue culture. Thus the polyhedrin open reading frame can be replaced by coding regions for target genes of choice, and in many cases, target proteins are expressed at levels equivalent to that of polyhedrin, which is approximately 1 mg per ml of culture. In addition, eukaryotic proteins are frequently subject to the appropriate post-translational modifications, including phosphorylation, glycosylation, and acylation.
Autographa californica nuclear polyhedrosis virus (AcNPV) is the prototype member of the Baculoviridae, which is a large family of DNA viruses that are pathogenic for invertebrates. The AcNPV genome consists of a double-stranded, supercoiled DNA molecule of 134 kbp, and potentially encodes 150 proteins (Ayres et al., 1994). In infected cells, AcNPV genes are expressed in a temporally controlled and ordered fashion (Blissard and Rohrmann 1990; O""Reilly et al., 1992). Viral genes are classified as early or late based on their requirements for viral DNA replication. Transient expression assays suggest that there are two distinct classes of early genes. One class includes genes like ie1 and ie2 that are highly expressed in the absence of other viral proteins and enhancer elements (Guarino and Summers, 1986a; Carson et al., 1988). The other class includes genes like 39k that are expressed at basal levels in the absence of viral factors, but whose expression is enhanced approximately 1000-fold in the presence of IE1 and cis-linked enhancer elements (Guarino and Summers, 1986b). Baculovirus early promoters resemble those transcribed by eukaryotic RNA polymerase II (pol II). Transcription of the early genes is inhibited by xcex1-amanitin, consistent with the hypothesis that early genes are transcribed by host pol II (Fuchs et al., 1983; Grula et al., 1981). Transcription of several early baculovirus genes initiates within a conserved xe2x80x98CAGTxe2x80x99 motif. Mutagenesis of this element was shown to affect transcription initiation in the 39k promoter (Guarino and Smith, 1992) and in the gp64 promoter (Blissard et al., 1992), suggesting that CAGT functions as an initiator element.
Late genes are also divided into two classes: the late genes, many of which encode viral structural proteins, and the very late genes, which are associated with the formation of viral occlusions. Transcription of both classes of late genes is resistant to (xcex1-amanitin (Huh and Waver, 1990), suggesting that these genes are transcribed by a viral-encoded RNA polymerase. This polymerase may be encoded by the lef-8 gene of AcNPV (Passarelli et al., 1994). Late and very late genes contain the consensus late promoter element, TAAG. This core element appears to function as both a promoter and an mRNA start site (Rankin et al., 1988).
Several proteins (LEF=late expression factor) required for late gene expression have been identified (Table 1; Passarelli and Miller, 1993a,b,c,1994; Passarelli et al., 1994; Li et al., 1993; Morris et al., 1994; Lu and Miller, 1994; McLachlin and Miller, 1994). The genes encoding these proteins were mapped using a transient transfection assay. Some of these proteins may be directly involved in late gene expression, and some may only be required for earlier events in the virus life cycle. For example, IE1 and IE2 transactivate early gene expression (Guarino and Summers, 1986a; Carson et al., 1988); and LEFs 1-3, helicase, and DNA polymerase are required for DNA replication (Kool et al., 1994). Little is known about the roles of LEFs. The predicted amino acid sequence of LEF-8 contains a motif that is conserved in RNA polymerases from various sources (Passarelli et al., 1994).
B. In Vitro Transcription of Capped RNAs
A number of research protocols have been developed that require milligram amounts of purified mRNAs. These RNAs are usually transcribed in vitro using plasmids or PCR fragments as templates. The most commonly used RNA polymerases for in vitro transcription are the enzymes encoded by the bacteriophages T3, T7, and SP6. These RNA polymerases are useful because they are highly active, very processive enzymes that recognize a specific promoter. In vitro transcription reactions with these enzymes are usually done as run-off assays. In this type of assay, the plasmid DNA is linearized prior to transcription, and so the 3xe2x80x2-end of the message is determined by the end of the DNA template. As a result, the transcripts produced are appropriate for translation in prokaryotic systems, but are not optimal for eukaryotic systems because they are not processed at the 5xe2x80x2 end with a 5xe2x80x2-methyl-7-guanosine cap and at the 3xe2x80x2 end with a poly(A) tail.
The most common use of in vitro transcribed RNAs is cell free translation, either in rabbit reticulocyte or in wheat germ extracts. Efficient in vitro translation of the RNAs requires the presence of a 5xe2x80x2-methyl-7-guanosine cap. The cap structure is important for binding of ribosomes to the RNA and also for message stability. Thus uncapped transcripts, which are the normal product of bacteriophage RNA polymerases, are not efficiently translated in eukaryotic cell free systems. Other protocols that require capped transcripts include microinjection of mRNAs into oocytes or transfection of mRNAs into animal cells or plant cells for the purpose of studying in vivo RNA processing, RNA transport, or protein function. Capping for these in vivo is essential because RNAs that are uncapped are rapidly degraded by cellular RNases. Also, in vitro splicing assays and the characterization of splicing factors require capped RNAs as substrate because the cap structure helps to target splicing enzymes to their substrates.
The production of capped mRNAs is usually done by adding cap analog (7mGpppG) to the in vitro transcription reactions. Cap analog can be incorporated in place of GTP al the 5xe2x80x2 end of the message. Although this is not the typical route of cap formation, the use of cap analog in transcription reactions yields RNAs with authentic and fully functional caps. There are, however, several disadvantages to the use of cap analog. First, cap analog is an expensive reagent, and costs approximately 20-times more than GTP. Second, the use of cap analog reduces the yields of RNA because the GTP concentration must be reduced to favor incorporation of the cap. Furthermore, even under these optimized conditions, only 80% of the messages are capped. For very long messages, the ratio of GTP to cap analog has to be increased to permit synthesis of full-length messages, thus further reducing the proportion of capped messages. Another problem is that cap analog binds to eukaryotic translation initiation factors and so is a competitive inhibitor of translation. This necessitates the complete removal of cap analog from the mRNA prior to translation, a step that could potentially lead to loss of RNA or other complications. Finally, it has been shown that methylated cap analogs are frequently incorporated into RNAs in a reverse orientation, so that the 5xe2x80x2 end of the message is Gppp7mGNNN instead of 7mGpppGNNN (Pasquinelli et al., 1995). The proportion of reverse-capped RNAs can be as high as 50%, and this is particularly problematical because many proteins that interact with mRNA caps do not recognize reverse caps.
The only commercially available alternative to cap analog is vaccinia virus capping enzyme. This two-subunit complex contains all three of the enzymatic activities (RNA 5xe2x80x2-triphposphatase, guanylyltransferase, and cap methyltransferase), that are required for the formation of a 5xe2x80x2 cap. Thus vaccinia virus capping enzyme can be used for posttranscriptional modifications of RNA after synthesis by one of the bacteriophage RNA polymerases. Caps can then be added using GTP and S-adenosyl-methionine (SAM). This is the normal route of synthesis, and these reagents are cheaper than cap analog. The drawback is that vaccinia capping enzyme is normally targeted to RNA substrates through interactions with vaccinia RNA polymerase, and in the absence the cognate RNA polymerase, very high molar amounts of capping enzyme have to be used to drive the capping reaction. Thus the advantage gained by avoiding the use of cap analog is more than offset by the expense of the capping enzyme.
C. In Vitro Transcription of Polyadenylated RNAs
The 3xe2x80x2 end of most eukaryotic mRNAs contains a stretch of adenylate residues, usually 100-200 nt in length. In vivo, the poly(A) tail has two primary effects. It increases message stability, and also binds factors that enhance translation initiation (Jackson and Standart, 1990). In vivo, the effects of capping and polyadenylation are synergistic so that the translational efficiency of capped and polyadenylaled messages is increased 450-fold compared unprocessed messages; capping alone increases translation only 20-fold, while polyadenylation alone has little effect (Gallie, 1991). Thus polyadenylation is highly recommended for RNAs to be used for transfection into eukaryotic cells.
The commercially available eukaryotic translation systems, wheat germ and rabbit reticulocyte, are relatively insensitive to the polyadenylation signals, however, and so 3xe2x80x2 processing is often omitted. Some companies (e.g. Promega) recommend it for optimal translational efficiencies because it does result in a 2-5-fold increase, lower than in vivo but still significant.
There are no commercially available enzymes for the production of polyadenylated RNAs, so messages with poly(A) tails are commonly produced using vectors that contain an oligo(dT) region on the template strand. The disadvantage of these vectors is that the poly(A) tail is usually shorter than normal (30 nt instead of 100-200), and there are frequently non-A residues at the end of the tail because it is necessary to have a restriction enzyme recognition site downstream of the templated adenylates.
Recently, two in vitro translation systems have been developed that better mimic the response of the eukaryotic translational machinery to the 5xe2x80x2 cap and poly(A) tails. In the yeast system, capped and polyadenylated mRNAs were translated 750-fold more efficiently than unprocessed RNAs, as compared to only a 3.7-fold increase for the same RNAs in reticulocyte extracts and a 6.8-fold increase in wheat germ extracts (Iizuka et al., 1994). A Drosophila embryo in vitro translation system, which similarly reproduces the synergism between the cap and poly(A) tail, has also been reported (Gebauer et al., 1998). The use of these systems promises to increase the power and utility of in vitro translation systems, but they can only be widely adapted by the research and diagnostic communities if convenient systems are simultaneously supplied for in vitro transcription of capped and polyadenylated RNAs.
The present invention solves these problems in the art by providing a baculovirus RNA polymerase that is relatively simple, being a four subunit complex, that can transcribe, cap and polyadenylate transcripts in vitro. Polyadenylation offers at least significant increases in stability of mRNAs and at least increases the efficiency of translation. The present invention hence solves the limitations in the prior art, including those addressed above. Present objects of the present invention therefore relate to materials and methods for producing stable, capped and polyadenylated RNAs in vitro and in vivo using a single RNA polymerase enzyme in methods that overcome prior art limitations.
The inventors have discovered that baculovirus RNA polymerase has the intrinsic ability to both cap and polyadenylate transcripts. The inventors presently understand that no other RNA polymerase is known with the intrinsic ability to both cap and polyadenylate transcripts. Prior to the discoveries of the present invention, most baculovirologists long assumed that polyadenylation was mediated by host enzymes. Indeed, showing this belief, most baculovirus expression vectors currently sold commercially have incorporated eukaryotic signals for cleavage and polyadenylation.
An embodiment disclosed herein provides a method of capping and polyadenylating RNA transcripts comprising the steps of: producing an RNA transcript using baculovirus RNA polymerase, wherein the baculovirus RNA polymerase caps and polyadenylates the RNA transcript.
In specific embodiments, producing the RNA transcript comprises an expression vector comprising a promoter sequence, a polynucleotide sequence and a baculovirus terminator sequence. The expression vector is prokaryotic or eukaryotic. More, particularly, the promoter sequence comprises a baculovirus consensus sequence or a sequence that is functionally equivalent. The consensus sequence is TAAG.
In further embodiments producing RNA transcripts may further comprise an accessory protein. These accessory proteins may enhance, for example, transcription, capping and/or polyadenylation. Exemplary accessory proteins include, but are not limited to, LEF-5 or VLF-1. Another accessory protein is a viral methyltransferase. Also, contemplated is that more than one accessory protein may be used to further enhance transcription.
In further embodiments, the method is performed in a cell. The cell is prokaryotic or eukaryotic. Yet further, the method is performed in a cell-free system or in vivo.
Specifically, the polymerase is a four subunit complex. The subunit complex comprises LEF-8, LEF-4, LEF-9 and p47. More particularly, LEF-4 subunit mediates capping. Yet further, polymerase terminates after a T-rich region in the transcript.
Also disclosed herein is a method of capping RNA transcripts comprising the steps of: producing an RNA transcript in vitro using baculovirus RNA polymerase, wherein the baculovirus RNA polymerase caps the RNA transcript. Specifically, the polymerase is a four subunit complex comprising LEF-8, LEF-4, LEF-9 and p47. hi specific embodiments, LEF-4 subunit mediates capping.
Another embodiment is a method of polyadenylating RNA transcripts comprising the steps of: producing an RNA transcript in vitro using baculovirus RNA polymerase, wherein the baculovirus RNA polymerase polyadenylates the RNA transcript. More specifically, the polymerase terminates after a T-rich region in the transcript.
In other specific embodiments, the present invention herein discloses an in vitro transcription kit comprising: a vector comprising a promoter sequence, a multiple cloning site and a baculovirus terminator sequence; transcription mix; and a baculovirus RNA polymerase. More particularly, the vector is prokaryotic or eukaryotic.
Yet further, the promoter comprises a baculovirus promoter consensus sequence or a functional equivalent of the baculovirus consensus sequence. The consensus sequence is TAAG. The polymerase is a four subunit complex. Specifically, the subunit complex comprises LEF-8, LEF-4, LEF-9 and p47. In specific embodiments, LEF-4 mediates capping.
In further embodiments, the kit may comprise an accessory protein. It is contemplated that an accessory protein enhances transcription. Exemplary accessory proteins include, but are not limited to, LEF-5, VLF-1 or a viral methyltransferase. It is also contemplated that more than one accessory protein may be used in the kit.
Another embodiment of the present invention is a coupled transcription-translation kit comprising: a vector comprising a promoter sequence, a multiple cloning site and a baculovirus terminator sequence; transcription mix; translation mix; and a baculovirus RNA polymerase. Specifically, the vector is prokaryotic or eukaryotic.
In specific embodiments, the promoter comprises a baculovirus promoter consensus sequence or a functional equivalent. More particularly, the consensus sequence is TAAG.
In a further specific embodiment, the polymerase is a four subunit complex. Specifically, the subunit complex comprises LEF-8, LEF-4, LEF-9 and p47. LEF-4 mediates capping.
In further embodiments, the kit may comprise an accessory protein. The accessory protein may be LEF-5, VLF-1 or a viral methyltransferase. Yet further, the kit may comprise more than one accessory protein.
As used herein the specification, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more. As used herein in the claim(s), when used in conjunction with the word xe2x80x9ccomprisingxe2x80x9d, the words, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more than one. As used herein xe2x80x9canotherxe2x80x9d may mean at least a second or more.
Other objects, features and advantages of the present invention are apparent in the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention are apparent to those skilled in the art in this detailed description.